In the relatively recent past, cell-free protein synthesis has become established as an efficient alternative to the expression of proteins in vivo (Carlson, E. D. et al., Biotechnology Advances, 2012, 30(5): pp. 1185-1194). Herein, the contents of the cell are used in order to manufacture a particular target protein in a rapid, reliable and cost-effective manner. The cell extracts obtained, also referred to as cell lysates, contain the essential components that are needed for the cell-free synthesis of proteins: ribosomes, translation factors and enzymes. Nowadays, selected recombinant proteins can be manufactured in a functionally active form in prokaryotic as well as in eukaryotic cell lysates. The following translation systems based on eukaryotic cell lysates are increasingly used currently: wheatgerm lysates, reticulocyte lysate, insect cell lysates and cell extracts from HeLa and HeLa hybridoma cells.
Compared with prokaryotic in vitro translation systems based on Escherichia coli, the protein yields achieved using most of the eukaryotic translation systems are relatively low (Carlson et al., above). An exception in this regard is the extremely efficient wheatgerm lysate expression system. Depending on the protein and reaction format, this achieves several hundred micrograms of protein per milliliter of reaction volume (Madin, K. et al., Proc. Natl. Acad. Sci. USA, 2000. 97(2): pp. 559-64). However, this cell extract is not suitable for synthesizing proteins with posttranslational modifications like, for example, glycosylations.
Eukaryotic translation systems based on insect cell lysates and reticulocyte lysates enable the synthesis of complex structured eukaryotic proteins with posttranslational modifications which cannot be synthesized in Escherichia coli. However, reticulocyte lysates must be enriched for this purpose with microsomal membranes of another species (heterogeneous translation system) In contrast thereto, cell lysates from Spodoptera frugiperda can be used as a homogeneous translation system, since the cell lysate and the membrane vesicles contained are obtained from the same cell line. By means of suitable cell decomposition methods, eukaryotic cell lysates can be obtained which contain components of an important cellular compartment, specifically the endoplasmic reticulum. All proteins that are discharged from the cell or are incorporated into the cell membrane, carry sugar residues or have disulphide bridges for stabilizing their molecular structure, migrate through this cellular compartment. Cell lysates which contain structures of the ER, so-called microsomes or membrane vesicles, can now be used to manufacture such protein candidates in a functionally active form. In this way, incompatibilities between the vesicles and the cytosolic proteins of the lysate are prevented, with the consequence of relatively high protein yields (up to 20 μg/ml in insect cell lysates in batch mode) and an efficient transport of target proteins into the microsomes of the lysate.
Cell-free protein synthesis reactions can be realized experimentally in a variety of ways. The simplest reaction route is the synthesis of a target protein in a one-pot synthesis (or batch reaction). Batch-based systems are therefore suitable for uncomplicated and rapid synthesis of a target protein. On the other hand, however, they are characterized by short run times and relatively low protein yields.
In general, a batch-based cell-free translation reaction reaches the maximum of synthesized target protein after 1-1.5 hours. Incubation times beyond this do not lead to increased protein yields, but result, with a high probability, in a reduction of the concentration of target protein, possibly due to the proteolytic decomposition of the target proteins, for example, by proteases present in the cell extract. However, it would be highly advantageous in particular cases also to preserve the synthesized target proteins over longer incubation times (>2 h) in an intact form in the translation solution.
One possibility for prolonging the run time of a cell-free protein synthesis reaction in order thereby to obtain greater protein yields is the use of dialysis systems (continuous exchange cell-free systems, CECF; Spirin, A. et al., Science, 1988, 242(4882): pp. 1162-4). Herein, energy-rich substances such as ATP and GTP pass by diffusion through a membrane into the reaction compartment, the site of the translation. At the same time, the reaction is depleted of inhibiting substances such as free phosphates and ADP.
The continuous supply of the cell-free reaction in the reaction compartment prolongs the run time of the synthesis and leads to significantly raised protein yields as compared with the batch system. Dialysis systems of this type are already commercially available, although exclusively in combination with prokaryotic cell lysates from Escherichia coli or with wheatgerm lysates.
Despite the yield increase as compared with discontinuous batch systems, the protein yield with corresponding eukaryotic dialysis systems is still relatively low compared with an Escherichia coli-based system.
Against this background, it is an object of the invention to provide means for enabling the synthesis of complex eukaryotic and prokaryotic proteins in a stable and functionally active form, particularly membrane proteins or proteins with posttranslational modifications in a relatively large quantity in a cell-free translation system.
This object is solved according to the invention by the method for cell-free protein synthesis according to the invention, wherein a eukaryotic cell lysate is used and the translation reaction is carried out in the presence of a caspase inhibitor, the use of a caspase inhibitor according to the invention, and the device for carrying out a method of the invention.